Extraction of Anthocyanins from Borage (Echium amoenum) Flowers Using Choline Chloride and a Glycerol-Based, Deep Eutectic Solvent: Optimization, Antioxidant Activity, and In Vitro Bioavailability

Borage flower (Echium amoenum), an annual herb native to the Mediterranean region, is an excellent source of anthocyanins and is widely used in various forms due to its biological activities. In the present study, a choline chloride and glycerol (CHGLY)-based natural deep eutectic solvent (NADES) was applied in order to extract the anthocyanins from borage flowers. The traditional solvents, including water, methanol, and ethanol, were used to evaluate the efficiency of CHGLY. The results showed that CHGLY was highly efficient compared to the traditional solvents, providing the highest amounts of the total anthocyanin content (TAC), total phenolic content (TPC), total flavonoid content (TFC), individual anthocyanins, and antioxidant activity (DPPH radical scavenging (DPPH) and ferric-reducing antioxidant power (FRAP) assays). The most dominant anthocyanin found in studied borage was cyanidin-3-glucoside, followed by cyanin chloride, cyanidin-3-rutinoside, and pelargonidin-3-glucoside. The bioavailability % was 71.86 ± 0.47%, 77.29 ± 0.57%, 80.22 ± 0.65%, and 90.95 ± 1.01% for cyanidin-3-glucoside, cyanidin-3-rutinoside, by pelargonidin-3-glucoside and cyanin chloride, respectively. However, cyanidin-3-glucoside was the anthocyanin compound showing the highest stability (99.11 ± 1.66%) in the gastrointestinal environment. These results suggested that choline chloride and glycerol-based NADES is not only an efficient, eco-friendly solvent for the extraction of anthocyanins but can also be used to increase the bioavailability of anthocyanins.


Introduction
Human health and well-being are closely linked to one's environment, diet, and overall lifestyle. Free radicals are associated with increased incidence of cardiovascular, pulmonary diseases, and many types of cancers [1]. Like reactive oxygen species (ROS) and reactive nitrogen species (RNS), free radicals can be produced in the organism as a by-product of metabolism or introduced from a number of exogenous sources (pollution, radiation, drugs, etc.) [2,3]. Free radicals can adversely affect many biological molecules (nucleic acids, proteins, and lipids), which alters the biological activities and results in increased oxidative stress. Consequently, they are involved directly or indirectly in the activation of diseases, such as diabetes, neurodegenerative disorders, respiratory diseases, cardiovascular diseases, along with other various diseases and cancers [2]. Through the ages, humans have believed in the positive effects associated with using a variety of herbs and foods for the treatment of certain illnesses. More recently, over the past few decades, researchers have begun to study the composition and purported effects of herbal treatments used in traditional medicine. addition, the extraction conditions with the prominent CHGLY were optimized using the central composite of Response Surface Methodology (RSM), and the in vitro bioavailability of the extract obtained at the optimum conditions was determined.

Results and Discussion
2.1. FTIR Spectra, Viscosity, pH, and Conductivity of CHGLY The FTIR spectrum of NADES obtained from choline and glycerol (CHGLY) is shown in Figure 1. The low intensity of OH stretching bands at wavenumber 3500-3200 cm −1 confirmed the presence of a low quantity of water in CHGLY [24,25]. During the CHGLY preparation, 20% of water was introduced to tailor the viscosity and to facilitate manipulation and enhance the extraction performance. The OH stretching vibration at the wavenumber of 3300-3100 cm −1 indicated the formation of hydrogen bonding between HBA and HBD [24,26]. The wavenumber at 3200-2932 cm −1 and 1645 cm −1 wavenumber referred to C-H stretching bands and C=C stretching vibrations, respectively. In addition, the wavenumbers 1500-600 cm −1 correspond to the C-O, CH, C-C, and OCO stretching, deformation, or bending vibrations [27]. Thus, the CHGLY can be assessed to be a homogenized NADES in which the chemical group tied different bunding networks.
using the central composite of Response Surface Methodology (RSM), and the in vitro bioavailability of the extract obtained at the optimum conditions was determined.

FTIR Spectra, Viscosity, pH, and Conductivity of CHGLY
The FTIR spectrum of NADES obtained from choline and glycerol (CHGLY) is shown in Figure 1. The low intensity of OH stretching bands at wavenumber 3500-3200 cm −1 confirmed the presence of a low quantity of water in CHGLY [24,25]. During the CHGLY preparation, 20% of water was introduced to tailor the viscosity and to facilitate manipulation and enhance the extraction performance. The OH stretching vibration at the wavenumber of 3300-3100 cm −1 indicated the formation of hydrogen bonding between HBA and HBD [24,26]. The wavenumber at 3200-2932 cm −1 and 1645 cm −1 wavenumber referred to C-H stretching bands and C=C stretching vibrations, respectively. In addition, the wavenumbers 1500-600 cm −1 correspond to the C-O, CH, C-C, and OCO stretching, deformation, or bending vibrations [27]. Thus, the CHGLY can be assessed to be a homogenized NADES in which the chemical group tied different bunding networks.
The viscosity, pH, and electric conductivity of CHGLY were found to be 22.89 ± 0.10 mPa, 5.03 ± 0.01, and 770.50 ± 10.25 µS.cm −1 , respectively. Viscosity is an important factor for the NADESs application in the extraction of bioactive compounds. NADES with high viscosity decreases the mass transfer in the extraction matrix [18]. In accordance with the viscosity found in the present study, Yadav et al. [28] have reported a viscosity of 21.37 mPa for the NADES prepared with choline chloride and glycerol at a 1:2 molar ratio and with the addition of 20% of water. Additionally, CHGLY has been reported as an adequate NADES for the extraction of phenolic compounds [29].  The viscosity, pH, and electric conductivity of CHGLY were found to be 22.89 ± 0.10 mPa, 5.03 ± 0.01, and 770.50 ± 10.25 µS.cm −1 , respectively. Viscosity is an important factor for the NADESs application in the extraction of bioactive compounds. NADES with high viscosity decreases the mass transfer in the extraction matrix [18]. In accordance with the viscosity found in the present study, Yadav et al. [28] have reported a viscosity of 21.37 mPa for the NADES prepared with choline chloride and glycerol at a 1:2 molar ratio and with the addition of 20% of water. Additionally, CHGLY has been reported as an adequate NADES for the extraction of phenolic compounds [29].
ANOVA obtained results are shown in Table 3. According to them, the quadratic model has been found suitable for the representation of experimental data. In general, and for all responses, the significance of the model was very low (p < 0.0001), and the insignificant lack-of-fit had high values (p > 0.1735). Additionally, the model had satisfactory R 2 (>0.9429) and adjusted-R 2 (>0.8762). All this confirms the closeness between experimental and the predicted values. After the model selection, the developed model terms of all the responses are shown in Table 3. Generally, temperature linear terms (X 3 ) were highly significant (p < 0.0001) for most responses, except for FRAP and cyanidin-3-rutinoside responses. According to the number of responses and their significance, the temperature was followed by water content (X 2 ), time (X 4 ), and finally by molar ratio linear terms; however, time quadratic terms (X 4×4 ) were highly significant (p < 0.0009) for all responses, excluding the cyanidin-3-rutinoside response. Molar ratio quadratic terms (X 1 X 1 ) exhibited significance for many responses, followed by temperature (X 3 X 3 ) and water content (X 2 X 2 ) quadratic terms. Moreover, terms of interactions have shown another tendency, where molar ratiotime (X 1 X 4 ) interactions had a high significance for most responses (p < 0.0343), followed by temperature-time (X 3 X 4 ), temperature-water content (X 2 X 3 ), and time-water content (X 2 X 4 ) interactions. The final polynomial equations are given in terms of the coded factors for all studied responses as follows:   Model and terms are significant at p ≤ 0.05.The previous equations are used to generate the perturbation plots (Figure 3), which helps to compare the effect of factors in one chosen point (A = 3.5, B = 30, C = 58, D = 25). A, C, B, and D represent molar ratio, water content, temperature, and time, respectively. For TAC (Figure 3a), factors, the molar ratio (A), temperature (C), and time (D) had a high extraction effect at their low and high limits and gave a weak extraction at the center point. However, water content factor (B) gave the lowest values in low limits, which means that the extraction efficiency of TAC was very weak at low water content, and after that, the effect was stabilized. Time seemed to be more effective more than other factors for long-duration treatment.
Moreover, TPC demonstrated another tendency (Figure 3b); the extraction of phenolic compounds has a positive relationship with water content and temperature; meanwhile, the molar ratio gives a high extraction efficiency when it approaches from the center point. Time presented more efficiency after 25 min of extraction. Similar to TPC, TFC extraction efficiency had a positive relationship with increasing temperature. FRAP analysis presented the effect of increasing water content and almost a stable effect of molar ratio and temperature variation. The antiradical scavenging capacity presented by DPPH (Figure 3e) has been affected; positively by increasing extraction temperature and negatively by increasing water content. Varying the molar ratio was more efficient around the midpoint. Beyond the center point, extraction time was more efficient at positive and negative extremes to give the highest values for TFC, FRAP, and DPPH. Model and terms are significant at p ≤ 0.05.The previous equations are used to generate the perturbation plots (Figure 3), which helps to compare the effect of factors in one chosen point (A = 3.5, B = 30, C = 58, D = 25). A, C, B, and D represent molar ratio, water content, temperature, and time, respectively. For TAC (Figure 3a), factors, the molar ratio (A), temperature (C), and time (D) had a high extraction effect at their low and high limits and gave a weak extraction at the center point. However, water content factor (B) gave the lowest values in low limits, which means that the extraction efficiency of TAC was very weak at low water content, and after that, the effect was stabilized. Time seemed to be more effective more than other factors for long-duration treatment.  Moreover, TPC demonstrated another tendency (Figure 3b); the extraction of phenolic compounds has a positive relationship with water content and temperature; meanwhile, the molar ratio gives a high extraction efficiency when it approaches from the center point. Time presented more efficiency after 25 min of extraction. Similar to TPC, TFC extraction efficiency had a positive relationship with increasing temperature. FRAP analysis presented the effect of increasing water content and almost a stable effect of molar ratio and temperature variation. The antiradical scavenging capacity presented by DPPH (Figure 3e) has been affected; positively by increasing extraction temperature and negatively by increasing water content. Varying the molar ratio was more efficient around the midpoint. Beyond the center point, extraction time was more efficient at positive and negative extremes to give the highest values for TFC, FRAP, and DPPH.
However, the long extraction time affected the content of most individual anthocyanins (Figure 3f-i). Unexpectedly, a short extraction time gave a high content of cyanidin chloride and cyanidin-3-glucoside, more than 750 mg/kg and 1000 mg/kg.b respectively. Meanwhile, for the same components, the high levels of molar ratio, water content, and  However, the long extraction time affected the content of most individual anthocyanins (Figure 3f-i). Unexpectedly, a short extraction time gave a high content of cyanidin chloride and cyanidin-3-glucoside, more than 750 mg/kg and 1000 mg/kg.b respectively. Meanwhile, for the same components, the high levels of molar ratio, water content, and temperature enhanced their extraction. For almost all factors, high levels negatively affected the extraction of cyanidin-3-rutinoside. The high molar ratio and extraction temperature drastically decreased the content of recovered pelargonidin-3-glucoside; in contrast, a short extraction time also gave weak levels.
The obtained results are consistent with a list of studies performed on the effect of extraction conditions using deep eutectic solvents. For instance, the study of de Almeida Pontes et al. [41] on olive leaves showed an improvement in the extraction of phenolic compounds by increasing extraction temperature (>50 • C). Additionally, it was demonstrated that variation in the amount and composition of deep eutectic solvents contents influences the composition and the quantity of recovery phenolic compounds. In addition, Cui et al. [42] found that the extraction yield of polyphenols was closely related to extractions parameters (time, temperature, liquid ratio, water content). Da Silva et al. [25] analyzed the effect of the molar ratio of choline-chloride: glycerol: citric acid mixture on the extraction of blueberry anthocyanins. They indicated the existence of a relationship between the anthocyanin's extraction efficiency and the chosen molar ratio. In the same way, Zannou et al. [21] observed that deep eutectic extraction behaves differently, and anthocyanins were sensible to all the studied factors (molar ratio, solvent ratio, and additional water).

Multi-Response Optimization on the Responses Using RSM
RSM was performed to identify the optimum conditions to obtain the maximum responses. The optimum conditions were determined by applying the desirability function, where the independent variables were kept in range, and the responses were maximized. The optimum conditions for maximum responses were a 1:4.62 molar ratio, 23.33% water content, a temperature of 74 • C, and 15 min extraction time. Under these optimum conditions, the predicted values of TAC, TPC, TFC, FRAP, DPPH, cyanin chloride, cyanidin-3-glucoside, cyanidin-3rutinoside, and pelargonidin-3-glucoside were 4.39 mg c3gE/g, 42 39 ± 0.97 mg/kg for TAC, TPC, TFC, FRAP, DPPH, cyanin chloride, cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-glucoside. The predicted and experimental data were found to be very close, which confirmed the reliability and reproducibility of the applied RSM process. As can be observed in Figure 4, the extract obtained in the optimum conditions had a high antioxidant activity, with the FRAP providing the highest value. Also. Figure 5 shows the chromatogram of individual anthocyanins obtained under optimal conditions.  TAC, TPC, TFC, FRAP, DPPH, cyanin chloride, cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-glucoside. The predicted and experimental data were found to be very close, which confirmed the reliability and reproducibility of the applied RSM process. As can be observed in Figure 4, the extract obtained in the optimum conditions had a high antioxidant activity, with the FRAP providing the highest value. Also. Figure 5 shows the chromatogram of individual anthocyanins obtained under optimal conditions.

In Vitro Bioavailability
An in vitro gastrointestinal model was applied to mimic the different steps of in vivo physiological digestion. Anthocyanins are the type of pH-sensitive phenolic compounds present in different chemical structures. The main chemical forms are flavylium cations in the stomach, while the carbinol forms predominate in the intestinal environment [43]. The bioavailability % and biostability % of the anthocyanin-enriched extract were investigated considering cyanin chloride, cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-glucoside ( Figure 6). The bioavailability of the evaluated anthocyanin compounds varied greatly in the simulated intestinal digestion (p ≤ 0.05). After the simulated intestinal digestion, cyanin chloride exhibited the highest bioavailability (90.95 ± 1.01%), followed by pelargonidin-3-glucoside (80.22 ± 0.65%), cyanidin-3-rutinoside (77.29 ± 0.57%), and cyanidin-3-glucoside (71.86 ± 0.47%), respectively. These findings were found close to the previous results of Mehran et al. [12], who determined a range of bioavailability of 70-90% for the anthocyanin extract of borage. Furthermore, Oliveira and Pintado [44] and Koh et al. [45] reported 88% and 90% bioavailability of cyanidin-3-glucoside after simulated intestinal digestion. Generally, anthocyanins are destroyed or biotransformed into other substances in the intestinal environment due to the high pH. Nonetheless, the bioavailability found in the present study was high and ranged from 70 to 90%, suggesting that CHGLY exerted a protective effect on borage anthocyanins. Anthocyanin compounds were less degraded due to the strong hydrogen bunding formed between CHGLY and anthocyanins. Similar to our findings, Da Silva et al. [46] found that the intestinal bioaccessibility of phenolic compounds was remarkably increased in the extract obtained from NADES (choline chloride:glycerol:citric acid; 0.5:2:0.5 molar ratio) compared to the extract obtained from conventional organic solvent (methanol:water:formic acid; 50:48.5:1.5; v/v/v), being about 35-fold higher for anthocyanins and 5-fold higher for non-anthocyanin phenolic compounds. Furthermore, Huang et al. [47] concluded that NADES is not only a sustainable ionic liquid with higher extraction efficiency but also an enhancer of oral bioavailability of specific natural products. The biotransformation of anthocyanins during the gastrointestinal tract changes greatly during the phase II metabolism processes and enzymatic and microbiota catabolism [43,48]. Di Lorenzo et al. [43] determined that the food matrix or technological/processing conditions, enzymatic patterns, and microbiota composition are the main factors affecting the bioavailability of anthocyanins in the gastrointestinal environment. As shown in Figure 6, the stability of the evaluated anthocyanins was found to be different in the intestinal environment. Although cyanidin-3-glucoside exhibited a low bioavailability compared to other anthocyanins, it presented the highest stability (99.11 ± 1.66%) in the intestinal environment. Cyanidin-3-glucoside was followed by cyanin chloride (96.37 ± 1.66%), pelargonidin-3-glucoside (93.39 ± 0.93%), and cyanidin-3-rutinoside (93.13 ± 1.96%).

Plant Material
Borage (Echium amoenum) flowers ( Figure 7) were collected in August 2021 from Oroumieh, Iran. The flowers were shade-dried for five days, sorted, and packed in brown bottles' screw caps.

Plant Material
Borage (Echium amoenum) flowers ( Figure 7) were collected in August 2021 from Oroumieh, Iran. The flowers were shade-dried for five days, sorted, and packed in brown bottles' screw caps.

Plant Material
Borage (Echium amoenum) flowers ( Figure 7) were collected in August 2021 from Oroumieh, Iran. The flowers were shade-dried for five days, sorted, and packed in brown bottles' screw caps.

Preparation of NADES
NADES was prepared according to Chanioti and Tzia [29]. HBA (Choline chloride) and HBD (glycerol) were combined at 1:2 molar ratio, followed by the addition of 20% of distilled water. Afterward, the mixture was heated for 2 h 30 min at 80 • C to obtain a homogenized liquid, briefly abbreviated CHGLY.

Physico-Chemical Characteristics of NADES
Viscosity of CHGLY was determined at 30 • C using a Rheometer (Buchi, CH-9230 Flawil, Switzerland) fitted with a parallel geometry with 20 mm of diameter and gap 1 mm [22]. pH was measured using a pH-meter (Model Starter 3100, OHAUS, Parsippany, NJ, USA). FTIR analysis of NADESs and extracts was carried out at the wavenumbers of 4000 and 400 cm −1 using an FTIR Spectrometer (Perkin Elmer, Spectrum-Two, Watham, MA, USA, PEService 35) [21]. Electrical conductivity properties were measured using an electrochemical analyzer (Consort, c6010, Turnhout, Belgium). The measurements were performed at 25 • C, and the values were recorded as µS.cm −1 .

Extraction of Anthocyanins
The extraction was carried out using a water bath. Distilled water, methanol, and ethanol (conventional solvents), and CHGLY (NADES), were used as solvents. A total of 0.3 g was mixed with 10 mL of solvents, and the mixture was ultrasonicated at 25 • C for 20 min. The samples were then filtered through Whatman filter paper No.1 thrice.

Total Phenolic Content (TPC)
TPC was evaluated by Folin-Ciocalteu method adopted from Nguyen et al. [49] with some modifications. Briefly, 150 µL of samples were mixed with 750 µL of 10% Folin-Ciocalteu reagent (5 min) and 600 µL of 7.5% Na 2 CO 3 . The mixture was kept in dark for 2 h, and the absorbance was read at 760 nm. TPC was expressed as mg gallic acid equivalent per g (mg GAE/g).

Total Flavonoid Content (TFC)
TFC was determined by adopting the procedure mentioned in Kim et al. [50]. The absorbance was read at 510 nm. The results were given as mg epicatechin equivalent per g (mg ECE/g).

Total Anthocyanin Content (TAC)
TAC was determined with the pH differential method reported in Lee et al. [51]. The absorbances of the samples containing pH 1 and pH 4.5 were read at 510 and 700 nm. TAC was expressed as mg cyanidin-3-glucoside equivalent per 100 g (mg CGE/100 g).
3.9. Determination of Antioxidant Activity 3.9.1. DPPH Radical Scavenging Activity Assay (DPPH) DPPH assay was conducted following the method of Pashazadeh et al. [52]. The absorbance was read against a control. The values of DPPH radical scavenging were determined with a calibration curve as mmol Trolox equivalent per g (mmol TE/g).

Ferric-Reducing Antioxidant Power Assay (FRAP)
FRAP assay was conducted following the method of Özdemir et al. [53]. The value of FRAP was obtained from a standard curve of FeSO 4 . The results were given as mmol FeSO 4 equivalents per g (mmol ISE/g).

Optimization with Response Surface Method (RSM)
The optimization parameters were examined systematically using response surface methodology based on the three-level central composite design (Design Expert software 13.0). The experimental design included four independent variables of X 1 (CHGLY, molar ratio), X 2 (water content, %), X 3 (temperature, • C), and X 4 (extraction time, min). The actual and coded values of the independent variables are shown in Table 4. The combination of parameters, such as molar ratio of CHGLY (1:0.5, 2, 3.5, 5, and 6.5), water content (10,20,30,40, and 50%), temperature (25,40,60,75, and 90 • C), and extraction time (5,15,25,35, and 45 min), were chosen as independent variables. From these variables, RSM generated 27 experimental points, including three replicates at the central point. TPC, TFC, TAC, DPPH radical scavenging activity, FRAP, and individual anthocyanin compounds were chosen as the responses (Y). The experimental points, together with responses, are given in Table 2. The analyses were performed in triplicate, and the results were given as means ± standard deviation. The experimental data were fitted to the following quadratic polynomial model: where Y is the response; X is the independent variable; β 0 is the model intercept coefficient; β i , β ii , β ij are interaction coefficients; k is the number of independent factors; and ź is the experimental error. The relationship between independent variables and responses was examined using analysis of variance (ANOVA) test in the Design Expert program.  ); (water content, %); X 2 (molar ratio) and X 3 (temperature, • C); X 4 (extraction time, min).

In Vitro Bioavailability
The in vitro bioaccessibility of anthocyanins was determined to be the fraction of anthocyanins that was solubilized within the mixed micelles and which became accessible for intestinal adsorption [55]. Following in vitro digestion, an aliquot of raw digesta was collected after the simulated small intestine digestion and centrifuged at 5000× g for 15 min at 4 • C. A supernatant (micelle fraction) was collected from the centrifuged digesta in which the anthocyanins were solubilized. A portion (3 mL) of the micelle fraction was vortexed after adding 3 mL of methanol and centrifuged at 5000× g for 15 min at 25 • C. The supernatant was then carefully collected and used for the determination of anthocyanins using HPLC-DAD. The bioaccessibility and stability of anthocyanins were then determined using the following equations: Bioaccessibility (%) = (C Micelle /C Digesta ) × 100 (11) Stability (%) = (C Digesta /C Initial ) × 100 (12) where C Initial , C Micelle , and C Digesta are the concentration of the individual anthocyanins initially, in the micelle phase, and the overall digesta at the end of the in vitro digestion, respectively.

Statistical Analyses
All results were expressed as the mean of three replicates ± standard deviation. Statistical analyses were performed using a one-way analysis of variance ANOVA, and the significance of the difference between means was evaluated by Turkey's test. Statistical significance was determined at p < 0.05. Design Expert software (version 13.0, Stat-Ease Inc., Minneapolis, MN, USA) was used for the RSM and experimental data analysis. ANOVA was used to determine the statistical relationship between factors. The adequacy of the models was determined by R 2 , adjusted R 2 , predicted R 2 , coefficient of variation (CV), adequate precision, p-value, and the value of Fisher's test (F-value). The significance of the models and regression coefficients were measured at p < 0.05. The behaviors of variables and responses were checked by the perturbation graphics. The optimum conditions were determined by applying the desirability function.